Abstract All organisms must maintain a balance between acquiring sufficient amounts of essential trace metals for growth and proliferation, and the toxicity associated with their elevated levels. Mammalian hosts use both copper limitation and copper overload as defense mechanisms against fungal pathogens. Fungi from the Pneumocystis genus cause pneumonia (PCP) in mammals with weakened immune systems. Mammalian lungs are a high copper environment, and these fungi maintain an extracellular life cycle in the lung alveoli which brings them into direct contact with an abundance of host factors including metals. In this pilot project, we seek to characterize molecular mechanisms underlying copper tolerance and homeostasis in Pneumocystis murina (Pm) that enable the fungus to counter the host Cu-driven cytotoxicity. The trace metal content was measured in Pm samples freshly extracted from mouse lungs with PCP infection, using size-exclusion chromatography (SEC). From a wide panel of measured metals, only Cu-, Fe-, and Zn-binding proteins of Pm showed pronounced distinct chromatographic profiles with elevated levels after accounting for the mouse metalloproteome background. Of the essential metals, copper is unique as it is required in trace amounts but quickly becomes highly toxic at elevated levels. The role of Cu in Pneumocystis biology is not known. Understanding Pm molecular mechanisms involved in Cu homeostasis will shed light on adaptation strategies of the fungus and may reveal new virulence factors and potential drug targets. The following specific aims are proposed: Aim 1. Identification of Cu-binding proteins ? to reveal (i) the Cu proteome of the host lungs infected with Pm and (ii) the primary Cu-binding proteins of the pathogen under pressure of excessive labile copper cytotoxicity. Cu-binding proteins will be separated and quantified using SEC-ICP-MS. Protein fractions from Pm and lung tissue will be enriched for Cu-proteins by immobilized metal-ion affinity chromatography (IMAC). Isolated proteins will be identified using shotgun proteomics (LC-MS/MS). Aim 2. Identification of Pm proteins induced by labile copper ? to identify the functional genes of the pathogen that orchestrate responses to excessive extracellular Cu. Gene induction by labile Cu2+ will be measured using RNA-seq following the addition of CuSO4 to the media using two concentrations, 1ng/ml and 100ng/ml, at 4-time points to reveal dynamic changes in gene expression. The project will advance knowledge on the Pneumocystis biology (Cu homeostasis) and shed light on the new axis of host-pathogen interactions for extracellular pathogens.